A series of substitution mutations affecting the Moloney murine leukemia virus MA protein were introduced into a cloned proviral DNA, and the mutant DNAs were tested for their biological activity in NIH/3T3 cells and COS cells. Many of the mutant viruses were viable and replicated with kinetics indistinguishable from the wild type. Seven mutants with alterations in a small region (residues 7-14 from the amino terminus) were replication-defective. These mutants were blocked in assembly and release of the virion particles in NIH/3T3 cells and were defective in both gag and gag-pol gene function. The results suggest that this very small region near the amino terminus of both proteins is required for their membrane targeting or self-association. Three of the defective mutant DNAs were able to induce virion particle formation when present at high copy number in COS cells.